Aging Selectively Reduces Mitochondrial Volume Density and Respiratory Capacity in Type IIx/IIb Diaphragm Muscle Fibers.

Diaphragm muscle (DIAm) sarcopenia involves selective atrophy of type IIx/IIb DIAm fibers. In addition, there is an age-related decrease in the maximum velocity of the succinate dehydrogenase reaction (SDH ) in type IIx/IIb fibers. SDH is a key enzyme of the tricarboxylic acid (TCA) cycle as well as complex II of the electron transport chain (ETC). These results suggest that sarcopenia may reduce mitochondrial volume density in type IIx/IIb DIAm fibers and thereby reduce SDH . Alternatively, sarcopenia may reduce the intrinsic respiratory capacity of mitochondria in type IIx/IIb DIAm fibers. We hypothesized that there is an age-related selective reduction in mitochondrial volume density and SDH per mitochondrial volume in type IIx/IIb DIAm fibers. In 24 Fischer 344 rats (6-mo: n=6 female, n=6 male; 24-mo: n=6 female, n=6 male), DIAm samples were dissected from the mid-costal region and rapidly frozen at optimal sarcomere length. In 10 mm cross-sections, DIAm fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Mitochondria were labelled using Mitotracker Green and imaged using 3D confocal microscopy (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. Each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI=SA /16µ V ) using ImageJ and NIS elements. In alternate 6 mm sections, SDH was measured using a quantitative histochemical assay in which accumulation of nitro blue tetrazolium diformazan (NBT ) was measured in DIAm fibers every 15 s for 10 min. Consistent with previous findings, type IIx/IIb DIAm fibers were ~36% smaller in 24-mo compared to 6-mo rats (P<0.0001), whereas there were no age-related differences in cross-sectional areas (CSA) of type I and IIa fibers. Mitochondrial volume density was lower in IIx/IIb fibers of 24-mo compared to 6-mo rats (P<0.0001), whereas there were no age-related differences in type I and IIa fibers. The MCI in type IIx/IIb fibers was lower (greater fragmentation) in 24-mo compared to 6-mo rats (P<0.0001), whereas the MCIs of type I and IIa fibers were similar across ages. Finally, SDH per fiber volume was lower in type IIx/IIb DIAm fibers in 24-mo compared with 6-mo rats (P<0.0001), whereas there were no age-related differences in type I and IIa fibers. When SDH was normalized for mitochondrial volume, there was no age-related difference in the intrinsic mitochondrial respiratory capacity in type IIx/IIb fibers. Therefore, aging results in reduced mitochondrial volume density and increased fragmentation in type IIx/IIb DIAm fibers, but no change in the intrinsic respiratory capacity of mitochondria.